Current Research Journal of Biological Sciences 1(3): 163-167, 2009
ISSN: 2041-0778
© M axwell Scientific Organization, 2009
Submitted Date: August 23, 2009
Accepted Date: September 02, 2009
Published Date: October 20, 2009
The Ability of Dermatophytes to Utilize Methylxanthine as
Sole Source of Carbon and Nitrogen
Ali A bdu l Hussein S. AL-Janabi
Department of Clinical Laboratory, College of Pharmacy, University of Karbala, Iraq
Abstract: Methylxanthine contains common compounds that widely consumed by human through their present
in food, drink and bevera ges or throug h invo lving in pharm aceu tical drug s. Dermato phytes were selected to
investigate for their ability to utilize three main compounds of methylxanthine as sole source of carbon and
nitrogen. Trichophyton mentagrophytes and Epidermophyton floccosum were grown on three types of media:
Medium A lacks of carbon source, medium B lacks of nitrogen source and medium C lacks of carbon and
nitrogen sources. Two isolated species of dermatophytes revealed well growth on medium A and less on med ia
B and C. Dermatophytes show ed ab ility to use methylxan thine as sole source of carbon and n itrogen with
efficiency to use carbon than nitrogen.
Key w ords: Caffeine, dermatophytes, methylxanthine, theophylline and theobromine
fermentation (SSF) in packed bed column bioreactor
(Tsubouchi et al., 1985). Furthermore, most of
Aspergillus ochraceus strains showed a significant degree
of degradation of caffeine w hen grow n on green coffee
med ium (T agliari et al., 2003).
On the other hand, many other species of fungi could
not be able to use methylxanthine as sole source of carbon
or nitrogen. Fusarium mo niliform e, Pen icillium
chrysogenum and the yeast Pichia butonii are good
examples for this inability when they could not utilize
caffeine and theobromine as sole source of nitrogen, w hile
other purine can be utilize by these fungi (Vega et al.,
2003; Allam and Elzainy, 1969; Allam and Elzainy,
1971).
Dermatophytes consider the most common
patho genic fungi causing cutaneous disease known as
dermatophytoses (Hainer, 2003). Infection by these fungi
is distributed all over the world. They contain only three
genera; Trichophyton, Microsporum and Epidermophyton.
Keratinous substances present in skin, hair and nail are
the prefer materials for nutrition of dermatophytes
(Simpan ya, 2000 ).
Determination the ability of dermatophytes to use
methylxanthine as sole source of carbon and nitrogen was
the aim of the present study.
INTRODUCTION
Methylxanthine is an importan t group of purin e
alkaloids. It consists of three main compounds; caffeine,
theophylline and theobromine (Scheind lin, 2007). Their
chemical structures are composed of nitrogen and carbon
within heterocyclic structure. Thus, methylxanthine can
be considered enrichment source of nitrogen and carbon
and degradation of its compounds can permit the
recycling of their elements into central metabolic pools of
environm ent (Zrenne r et al., 2006 ).
In plants-containing me thylxanthine w hich are
including Coffea arba cia, Cola nitida, Camellia sinensis,
catabolism of caffeine produces theophylline,
theophylline converts to 3- methylxanthine and
theobromine conv erts to caffeine (Ash ihara et al., 1996).
This degra dation proce ss is related to seasonal variation
(Mohanpuria et al., 2009 ).
Generally, caffeine degrading microbes have been
isolated from natural habitudes such as soil under coffee
cultivation and human domestic waste water (YamaokaYano and Mazzafera, 1998; Mohapatra et al., 2006).
Microorganisms, especially ba cteria an d fungi, play an
important role in degradation of methylxan thine to their
simple metabolites. In contrast with bacteria, a few
studies illustrated the ability of fungi to degrade
methylxanthine. Most of the m are focused on the activity
of fungi to catalyze caffeine than other methylxanthine
which may be related to highest available amounts of
caffeine in nature com pared w ith other members of
methylxanthine group. However, some fungi have the
ability to degrade methylxanthine while other are not
based on specific enzymes that fungi may have to perform
this process. The fungus Pleurotus florida can grow on
coffee wastes contain 3% caffeine and after fung al grow th
the percentage of caffeine decreased to 0.4% (Murthy and
Manonmani, 2008). Rhizopus delemar also showed ab ility
to catalyze caffeine and theop hylline by solid-state
MATERIALS AND METHODS
Organ isms: Two strains of dermatophytes including
Trichophyton mentagrophytes and Epidermophyton
floccosum were clinical isolated from AL-Hussein general
hospital of Karbala providence in February 2009. Skin
scales of fungal lesion w ere cultured on Sabouraud's
glucose agar of the following com ponents; glucose 20 g,
peptone 10 g, agar 15 g, chloramphenicol 0.05 g and
1000 ml of distilled water. Cultures were incubated at 28
ºC for two weeks. Grown fungi were diagnosed according
to criteria recorded by Rippon (1988) and Emmons
(1970).
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Chem ical agents: Theophylline, caffeine, and
theobromine were purc hased from HiM edia, M umb aiIndia. Different concentrations of m ethylxanthine were
used.
M edia preparation: Standard m edium u sed for ordinary
culturing of fungi contain d extrose or glucose as carbon
source and peptone as nitrogen source (Emmons, 1970).
Thus, three types of med ia we re prep ared that have the
following design: Medium A prepared from mixing
peptone 10 g, agar 15 g with 1 L of distilled water;
Medium B contain s glucose 20 g, agar 15 g dissolv ed in
1 L of distilled water; and medium C that contains agar 15
g only in 1 L of distilled water.
Fungal growth assay: Colony diameter method
employed by Kücüc and Kivan (2003) was used. Various
concentrations of methylxanthine were mingled w ith
melting prepared m edia. Then , poured in steriled Petri
dishes. A disk (9 mm) of old grown fungi (at 28ºC for 1
week) was inoculated on the center of culture media.
Plates were incubated at 28ºC for 1 week. Perpendicular
colony diameters (mm) of grown strains were measured.
Each experiment was repeated triplicate for statistical
analysis.
Fig. 1: Colonies diameter of grown T. mentagrophytes on
medium A (no carbon source)
Statistical analysis: Result data w ere statistically
analyzed by using two-way variance of analysis
(ANOVA) with less significant diffe rence (L.S.D .) at P <
0.05.
RESULTS
Fig. 2: Colonies diameter of grown E. floccosum on medium A
(no carbon source)
Caffeine, theophylline and theobromine are the main
mem bers of methylxanthine were selec ted for their ability
to supp ly dermatophytes by nitrogen and carbon when the
absence of other sources.
In the absence of glucose in medium A, distinct
grow th was observed of two strains of dermatophytes.
Lower concentrations of methylxanthine sho wed more
effectiveness to enhanc e fungal grow th. The grow th of T.
mentagrophytes regarded to be very well on medium A
than those o f E. floccosum (Fig. 1 and 2).
On media B and C, the density of fungal mycelia was
very low and may reach to form tiny extended colonies
(Fig. 3). Colony diameter of grown fungi was
approxim ately the same as on medium A (Fig. 4 and 5).
E. floccosum did not exhibit any growth on both B and C
med ia compared with enlargement diameter of T.
mentagrophytes colonies. However, lower concentrations
of methylxanthine also encouraged T. mentagrophytes to
grow well than at high doses of methylxanthine.
Fig. 3: Colony of T. mentagrophytes grown on medium B and
C
carbon and nitrogen by Serratia marcescens, paraxanthin
and or theobromine were released in liquid medium
(Ma zzafera et al., 1996). Fungi strains with the highest
ability to degrade caffeine were identified as Aspergillus
and Penicillium (Mazzafera, 2002). Hence, seven of
twenty strains of Aspergillus and Penicillium genuses
were able to grow on m edia containing caffeine (Hakil
et al., 1998 ).
Growth of dermatophytes on media containing
methylxanthine with lack of simple sugar as source of
carbon found to be excellent and combined with normal
DISCUSSION
Although bacteria consider the most effective
microorganisms of methylxanthine degradation that can
utilize the metabolites of methylxanthine as main source
of carbon and nitrogen, fungi also demonstrated to have
such ability. When caffeine was used as sole source of
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Curr. Res. J. Biol. Sci., 1(3): 163-167, 2009
only in pharmaceutical therapy, but in food, drink and
beverages (Chou, 19 92) C atabo lism of caffeine in
microorganisms commences via two possible
mechanisms; demethylation and oxidation. Through the
demethylation route, the major metabolite form in fungi
is theop hylline (Hakil et al., 1998), while theobromine is
the major metabolite in bacteria (Yamaoka-Yano and
Mazzafera, 1999). Enzymatic activities are considered
respo nsible of methylxanthine degra dation.
In bacteria, the conversion of caffeine to its
metabolites is primarily brought about by N-demethylase,
caffeine oxidase and xanthine oxidase that are produced
by several caffeine-degrading bacteria species such as
Pseudomonas putida and species within the genera
Alcaligenes, Rhodococcus and Kleb siella (Dash
and Gum madi, 200 6). W hereas, fungi caffeine in can
degrade by caffeinases en zyme s. These enzym es are
methyl-releasing from caffeine molecule to give
theophylline and methanol as products, then theophylline
is transformed in 3-methylxanthine and xanthine (Aguilar
et al., 2008). Thus, the principle caffeine degradation
prod ucts in fungi were theophylline and 3methylxan thine (T agliari et al., 2003 ).
In present study, dermatophytes did not prefer any of
methylxanthine than other to grow with. Ho wev er,
theophylline considers a complex compound for
microorganism 's nutrition and they co uld no t be able to
degrade it comp ared w ith other members of
methylxanthine. Grow th of Serratia marcescens was
diminished in the present of theophylline (Mazz afera
et al., 1996). All of twenty strains of Aspergillus and
Penicillium showed the efficiency to catalyze
theobromine with inability to degrad e theophylline (Hakil
et al., 1998).
In hum an bo dy, caffeine and other methylxanthine
are degradation in liver through the activity of cytochrom
P450 (Berthou et al., 1992). Sauer (Sauer, 1982) obtained
indications that caffeine in yeast was degraded by
cytochrom P450 suggesting that the catabolic pathway
might be similar to anim als. However, kinetic study
showed that caffeine-degradation by Aspergillus sp. was
related to the develo pme nt of mold and its respiration
(Brand et al., 2002).
Methylxanthine could be inhibited fungal grow th
when they presence in high concentrations. Jayaratna
et al. (2007) found that colony diam eter of
Monacrosporium ambrosium fungus grown on caffeine
containing media was significantly less than on the
control media. Fu rthermore, conidia germination of
Sporothrix schenckii was prevented by caffeine
(Rodriguez-Del-Valle et al., 1984). Thus, the results of
above studies supporting our finding, when low
concentrations of methylxanthine encouraged the growth
of dermatophytes more than at higher concentrations.
Additionally, development of a process involving
microbial enzymatic degradation of caffeine to non-toxic
compound is necessary to solve the problems of chemical
extraction of caffeine in food products as well as treating
the caffeine containing waste product (Gokulakrishnan
et al., 2005).
Fig. 4: Colonies diameter of grown T. mentagrophytes on
medium B (no nitrogen source)
Fig. 5: Colonies diameter of grown T. mentagrophytes on
medium C ( no carbon and nitrogen source)
density of fungal colonies. Based on these results, all of
three selected methylxanthine w ere consider suitable
source of carbon with sufficient supplying during the long
periods of incubation for dermatophytes growth. The
efficiency of dermatophytes for obtaining nitrogen from
methylxanthine degradation in the absence of peptone was
less than when fungi used methylxan thine to obtain
carbon. Thin den sity of T. mentagrophytes myc elia with
absent of E. floccosum grow th may consider an indicator
for ineffic i e n cy o f derm atoph ytes to u tilize
methylxanthine as nitrogen source with species-dependent
mann er. Thus, methylxanthine can be represented a good
source of carbon than nitrogen for dermatophytes growth.
By contrast, man y studies demonstrated that
methylxanthine can be useful for fungi as a source of
nitrogen when cu lturing m edia supplied with carbon
source. Results in H akill et al. (1999) study confirmed
that caffeine can be used in solid-state fermentation as
sole nitrogen source if sucrose is used as the carbon
source. The degradation of caffeine by Penicillium
verrucosum was complete (100%) at 72 h fermentation in
the absence of any added nitrogen source (Roussos et al.,
1994).
Caffeine is the most important agent of
methylxanthine group due to high consuming of it not
165
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less mycelia density on medium lack of peptone.
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